electrochemical detection of peroxynitrite using a biosensor...

Electrochemical Detection of Peroxynitrite Using aBiosensor Based on a ConductingPolymer-Manganese Ion ComplexWei Choon Alvin Koh,† Jung Ik Son,† Eun Sang Choe,‡ and Yoon-Bo Shim*,†

Department of Chemistry, Institute of BioPhysio Sensor Technology and Department of Biological Sciences, PusanNational University, Busan 609-735, Korea

A peroxynitrite (ONOO-) biosensor has been developedthrough the preparation of a new manganese-[poly-2,5-di-(2-thienyl)-1H-pyrrole)-1-(p-benzoicacid)](Mn-pDPB)complex. DPB monomer was first synthesized andpolymerized for the purpose of providing a polymerbackbone for complex formation with Mn2+ ion. TheMn-pDPB complex was characterized via Magneto-motive Force (MMF) simulation, X-ray photoelectronspectroscopy (XPS), and cyclic voltammetry. The com-plex selectively enhanced the reduction process ofONOO- which was used as the analytical signal forchronoamperometric detection. A polyethyleneimmine(PEI) layer was coated on the complex surface toincrease selectivity and stability. The chronoampero-metric calibration plot showed the hydrodynamic rangeof 2.0 × 10-8-5.0 × 10-7 M. The detection limit wasdetermined to be 1.9 ((0.2) × 10-9 M based on S/N) 3. The microbiosensor, fabricated on a 100 µmdiameter Pt tip, was applied in a real rat plasmasample for the detection of spiked concentrations ofONOO-. The reliability and long-term stability of themicrobiosensor was also examined with YPEN-1 cellsin vitro, and the results shown were promising.

Peroxynitrite (ONOO-) is a very powerful oxidant andcytotoxic agent produced in biological systems by the recom-bination of nitric oxide and superoxide anion radical. ONOO-

has been a source of both exciting discovery and vibrant debatewithin the broad community of chemically oriented biologists.Because of the reaction rate of its ubiquitous precursors, onecan expect to always contend with the direct and secondaryreactions of ONOO-. Research in this area has solidly estab-lished the contribution of ONOO- to the fundamental regula-tion of redox-dependent cell signaling,1 hemostasis,2 and hostdefense.3 Also, when xenobiotic exposure and inflammatoryresponses accelerate the generation of superoxide and nitricoxide, ONOO- further contributes to autoimmune, neurode-generative, apoptotic, genotoxic, and an abundance of target

molecule reactions that affect all aspects of tissue and cellularexistence.4-6 While stabilized as an anion at high pH, ONOO-

has a relatively short half-life (∼1 s) under physiologicalconditions due to rapid reaction with biological targets andmolecular decomposition via rearrangement or hemolyticscission.7 The most useful markers for ONOO- formation inthis context are nitration and hydroxylation products and thedimerization of tyrosine residues.8

The use of synthetic ONOO- in model systems and therigorous use of controls in biological systems (e.g., ONOO-

scavengers and suppression of superoxide and nitric oxideconcentrations) have provided a solid foundation of knowledgethat encourages the significance of this species as a dynamicredox signaling mediator and, at higher rates of production, atoxicant.9 Thus, it is important to quantify the details of ONOO-

production in biological tissues, including direct measurement.For detection of ONOO-, a variety of sensor systems has beendeveloped. Mass spectrometric and immunodetection of nitro-tyrosine is typically applied for the presence of biologicalONOO- formation.8,10 Other methods have also been developedfor the detection of ONOO-, such as UV-visible spectroscopy,electron spin resonance spectroscopy, chemiluminescence, andfluorescence.11-15 These analytical techniques allow the specificdetermination of ONOO-, but they are complicated, time-consuming, and require costly equipment. Otherwise, electro-chemical methods are most advantageous because of theirsimplicity, speed, and sensitivity as well as being able toperform measurements due to miniaturization of sensor ele-ments.16 Disadvantages of electrochemical methods includefouling of the biosensor surface and low selectivity. To

* Corresponding author. Phone:(+82) 51 510 2244. Fax: (+82) 51 514 2430.E-mail: [email protected].

† Department of Chemistry, Institute of BioPhysio Sensor Technology.‡ Department of Biological Sciences.

(1) Tarpey, M. M.; Fridovich, I. Circ. Res. 2001, 89, 224–236.(2) Eaton, P.; Clements-Jewery, H. Br. J. Pharmacol. 2008, 155, 972–973.(3) Habib, S.; Moinuddin; Ali, A.; Ali, R. Cell. Immunol. 2009, 254, 117–123.

(4) Woodcock, S. R.; Freeman, B. A. Chem. Res. Toxicol. 2008, 21, 2227–2228.(5) Starodubtseva, M. N.; Tattersall, A. L.; Kuznetsova, T. G.; Yegorenkov, N. I.;

Ellory, J. C. Biochemistry 2008, 73, 155–162.(6) Arbault, S.; Sojic, N.; Bruce, D.; Amatore, C.; Sarasin, A.; Vuillaume, M.

Carcinogenesis 2004, 25, 509–515.(7) Beckman, J. S.; Koppenolm, W. H. Am. J. Physiol. 1996, 271, 1424–1437.(8) Kirsch, C.; de Groot, H. J. Biol. Chem. 2002, 277, 13379–13388.(9) Virag, L.; Szabo, E.; Gergely, P.; Szabo, C. Toxicol. Lett. 2003, 140, 113–

124.(10) Latal, P.; Kissner, R.; Bohle, D. S.; Koppenol, W. H. Inorg. Chem. 2004,

43, 6519–6521.(11) Malinski, T.; Taha, Z. Nature 1992, 358 (6388), 676–678.(12) Kulagina, N. V.; Zigmond, M. J.; Michael, A. C. Neuroscience 2001, 102,

121–128.(13) Boon, E. M.; Marletta, M. A. J. Am. Chem. Soc. 2006, 128, 10022–10023.(14) Fabre, B.; Burlet, S.; Cespuglio, R.; Bidan, G. J. Electroanal. Chem. 1997,

426, 75–83.(15) Lee, Y. T.; Shim, Y. B. Anal. Chem. 2001, 73, 5629–5632.

Anal. Chem. 2010, 82, 10075–10082

10.1021/ac102041u 2010 American Chemical Society 10075Analytical Chemistry, Vol. 82, No. 24, December 15, 2010Published on Web 11/19/2010

overcome the shortage of these methods, we synthesized anew polymer-Mn2+ complex to improve selectivity and exam-ined the reliability of the sensor in ONOO- detection. The Mn2+

ion can enhance the electron transfer reaction involved in thedegradation of ONOO- to nitrogen dioxide and nitrate.17-19

Thus, we tried to utilize this reaction for the detection of ONOO-.Conducting polymers, having carboxylic acid as a functional

group, can coordinate with a metal ion to form a coordinationcomplex. Electropolymerization provides one of the in situ sensorpreparation methods, examples of sensory material being conduct-ing polymers such as polypyrrole14 and polyterthiophene.15 Thesefilms can be prepared reproducibly and quite thinly, ensuring arapid and stable response of the sensor.20,21 The organic functionalgroups, such as amine, imine, and carboxylic acid can be used asligands for the metal ion complexation.22,23 So far, there are fewreports of the metal ion complex with conducting polymers dueto the weak interaction of conducting polymers with metal ions.24

In the present study, in order to more flexibly control theorientation of the carboxylic acid groups outward from the probesurface, we have synthesized a new ligand, 2,5-di-(2-thienyl)-1H-pyrrole derivative, [(2,5-di-(2-thienyl)-1H-pyrrole)-1-(p-benzoic acid)](DPB). We also studied the preparation and characterization ofthe Mn-conducting polymer complex (Mn-pDTB)-coated mi-croelectrode and its electrocatalytic activity toward ONOO-

reduction. A polyethyleneimmine (PEI) layer was coated ontothe modified electrode surface to increase ONOO- selectivityand biosensor stability. The experimental parameters such aspH and applied potential were optimized. We demonstrated thebiosensor’s applicability to the in vitro determination of ONOO-

in a real plasma sample. In addition, the ONOO- biosensorwas also applied to stimulated cultured cells, and the validityof the sensor was evaluated.

EXPERIMENTAL SECTIONMaterials. A ter-heteroaromatic (thiophene-pyrrole-thiophene)

functionalized monomer, 2,5-di(2-thienyl)-1H-pyrrole-1-(p-benzoicacid) (DPB) was newly synthesized through the Paal-Knorrpyrrole condensation reaction.25 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), polyethyleneimmine (PEI), dichloromethane(CH2Cl2; 99.8%, anhydrous, sealed under nitrogen gas), hydro-gen peroxide (30% solution), manganese sulfate, and cocainehydrochloride were purchased from Sigma Aldrich (USA).Tetrabutylammonium perchlorate (TBAP, electrochemical grade)

was received from Fluka (USA), purified, and then dried undervacuum at 1.33 × 103 Pa. Disodium hydrogen phosphate,sodium dihydrogen phosphate, sodium chloride, sulfuric acid,and ethanol were purchased from Aldrich Chemical Co. (USA).A phosphate buffer saline solution (PBS) was prepared bymodifying 0.1 M of disodium hydrogen phosphate and 0.1 Mof sodium dihydrogen phosphate with 0.1% sodium chloride.All other chemicals were of extra pure analytical grade andused without further purification. All aqueous solutions wereprepared in doubly distilled water, which was obtained from aMilli-Q water purifying system (18 MΩ cm).

Preparation of Peroxynitrite Standard Solutions. ONOO-

was biomimetically synthesized from nitric oxide (NO)26 andpotassium superoxide27 solutions. ONOO- was also introducedvia a 0.1 mM donor solution of 3-morpholinosydnonimine (SIN-1).28 The ONOO- stock solution was stored at -20 °C, andthe concentration was verified by UV-visible spectrometry at302 nm (ε ) 1670 mol-1 L cm-1) just before the experiments.29

Microelectrode Preparation. The Pt microelectrode wasfabricated and subsequently cleaned by cycling the appliedpotential between +1.4 and -0.2 V for ten cycles at a scan rate of500 mV/s in a 0.5 M H2SO4 solution followed by washing withdistilled water. It was then used in all subsequent experimentsaccording to our previous report.30

Peroxynitrite Sensor Fabrication. The Mn-pDPB complex-ing solution was composed of 1.0 mM Mn2+ and 1.0 mM DPBmonomer together in a 0.1 M TBAP/CH2Cl2 solution. Elec-tropolymerization on the microelectrode surface was performedby cycling the potential between 0 and 1.4 V two times at thescan rate of 100 mV/s. After that, the electrode was washedwith CH2Cl2 to remove the excess monomer. Gold nanopar-ticles (AuNPs) were then electrodeposited on the modifiedelectrode surface using linear sweep voltammetry. PEI coatingwas performed by dipping the Mn-pDPB complex-modifiedelectrode three times in a 1% PEI solution. The modifiedelectrode was completely dried after PEI coating.

Instruments. A Mn-pDPB complex-modified microelectrode,Ag/AgCl (in saturated KCl), and a Pt wire were used as working,reference, and counter electrodes, respectively. Cyclic voltammo-grams and chronoamperograms were recorded using a poten-tiostat/galvanostat, Kosentech Model KST-P2 (South Korea).Electron spectroscopy for chemical analysis (ESCA) experimentswere done using a VG Scientific ESCALAB 250 XPS spectrometerwith a monochromated Al KR source and charge compensation(Korea Basic Science Institute, Busan). ChemDraw Ultra andMM2 software were used for 3D structure stimulation and bindingenergy calculation as shown in Figure 1c.

Electrochemical Measurements. Cyclic voltammogramswere recorded for the Mn-pDPB microelectrode from -0.2 to0.6 V versus Ag/AgCl in 0.1 M PBS at pH 7.4. Chronoampero-metric experiments were performed by applying the potential of0.2 V at the Mn-pDPB microelectrode to reduce ONOO-. A

(16) Yang, D.; Wang, H.-L.; Sun, Z.-N.; Chung, N.-W.; Shen, J.-G. J. Am. Chem.Soc. 2006, 128, 6004–6005.

(17) Xue, J.; Ying, X.; Chen, J.; Xian, Y.; Jin, L. Anal. Chem. 2000, 72, 5313–5321.

(18) Cortes, J. S.; Granados, S. G.; Ordaz, A. A.; Jimenez, J. A. L.; Griveau, S.;Bedioui, F. Electroanalysis 2007, 1, 61–64.

(19) Viggiano, A. A.; Midey, A. J.; Ehlerding, A. Int. J. Mass Spectrom. 2006,255, 65–70.

(20) Rahman, M. A.; Kwon, N.-H.; Won, M. S.; Choe, E. S.; Shim, Y.-B. Anal.Chem. 2005, 77, 4854–4860.

(21) Rahman, M. A.; Park, D.-S.; Chang, S. C.; McNeil, C. J.; Shim, Y.-B. Biosens.Bioelectron. 2006, 21, 1116–1124.

(22) Cotton, F. A.; Wilkinson, G. Advanced Inorganic Chemistry; John Wiley &Sons: New York, 1988.

(23) Mehrotra, R. C.; Bohra, R. Metal Carboxylates; Academic Press: London,1983.

(24) Rahman, M. A.; Lee, K.-S.; Park, D.-S.; Won, M.-S.; Shim, Y.-B. Biosens.Bioelectron. 2008, 23, 857–864.

(25) Amarnath, V.; Anthony, D. C.; Amarnath, K.; Valentine, W. M.; Wetterau,L. A.; Graham, D. G. J. Org. Chem. 1991, 56, 6924–6931.

(26) Pallini, M.; Curulli, A.; Amine, A.; Palleschi, G. Electroanalysis 1998, 10,1010–1016.

(27) Ge, B.; Lisdat, F. Anal. Chim. Acta 2002, 454, 53–64.(28) Ashki, N.; Hayes, K. C.; Bao, F. Neuroscience 2008, 156, 107–117.(29) Vander, V. A.; Eiserich, J. P.; O’Neill, C. A. Arch. Biochem. Biophys. 1995,

319, 341–349.(30) Koh, W. C. A.; Rahman, M. A.; Choe, E. S.; Lee, D. K.; Shim, Y.-B. Biosens.

Bioelectron. 2008, 23, 1374–1381.

10076 Analytical Chemistry, Vol. 82, No. 24, December 15, 2010

freshly prepared 4.0 mL aliquot of 0.1 M PBS was added intothe electrochemical cell, and the steady-state current wasmonitored with the Mn-pDPB microelectrode at the optimalpH and temperature. Consecutive injections of varying amountsof ONOO- into the cell and their amperometric responses weremonitored. In in vitro experiments, there was a three-electrodeconfiguration where the ONOO- microbiosensor, Ag/AgClelectrode, and Pt wire were used as the working, reference,and counter electrodes, respectively. All biosensors werecalibrated at 25 ± 1 °C.

Blood Plasma Sample. The rat blood plasma real samplewas prepared according to the following procedure; at first, 2 µg/mL heparin was added to the rat blood samples to preventcoagulation. The blood plasma was then centrifuged for 15 minat 4000 rpm. The liquid was then centrifuged twice at 10 000 rpmfor 15 min each time before the experiments.

Cell Culture Sample. YPEN-1 glioma cells (American TypeCulture Collection, anassas, VA) were cultured in Dulbecco’sModified Eagle’s Medium (Gibco), supplemented with 15% fetalcalf serum (Gibco), 0.1 mM mercaptoethanol (Sigma), 0.1 mMnonessential amino acids (Gibco), 100 U/mL penicillin, and 100mg/mL streptomycin (Gibco). Briefly, cells were trypsinized andsuspended in 10 mL of differentiation medium (Iscove’s ModifiedDulbecco’s Media), 15% FBS, 2.0 mM L-glutamine, 0.1 mM

nonessential amino acids, 100 U/mL penicillin, and 100 mg/mLstreptomycin and cultured in 100 mm nonadhesive Petri dishesto allow cells to aggregate and form embryoid bodies (EBs). Themedium was replaced every 2 days. Cells were plated on 0.1%gelatin coated Petri dishes.

RESULTS AND DISCUSSION

Preparation and Characterization of the Mn-pDPB Mi-croelectrode. The schematic representation of the preparationof the Mn-pDPB complex on the electrode is shown in Scheme1. The nanoparticle comprised conducting polymer layer onto amicroelectrode was obtained through the electropolymerizationreaction of 1.0 mM DPB monomer containing 1.0 mM Mn2+ in a0.1 M TBAP/CH2Cl2 solution by cycling the potential between0 and 1.4 V two times at the scan rate of 100 mV/s. As shownin Figure 1a, an increasing reduction and oxidation peak for DPBwith each potential cycle was obtained at 570 and 950 mV,respectively, after the oxidation of monomer at +900 mV. Redoxpeaks for Mn2+ ion were not clearly shown because DPB hasredox peaks of a much higher magnitude than Mn2+ due tothe relatively low electroactivity of Mn2+ in nonaqueous

Figure 1. (a) Cyclic voltammograms recorded for the electropolymerization of DPB monomer in a 0.1 M TBAP/CH2Cl2 for three consecutivepotential cycles. (b) CVs of the Mn-pDPB complex-modified surface without peroxynitrite (0 µM), in the presence of peroxynitrite (46 µM), andonly on the pDPB surface. (c) 3D image stimulation and calculated MMF energies of Mn-pDPB structure.

10077Analytical Chemistry, Vol. 82, No. 24, December 15, 2010

solution.31 After that, the electrode was washed with CH2Cl2to remove the excess monomer. Gold nanoparticles (AuNPs)were then electrodeposited on the modified electrode surface.The Mn-pDPB modified electrode was coated with a film ofPEI. The modified electrode was completely dried after PEIcoating. Figure 1b shows the CVs recorded for a Mn-pDPBcomplex-modified electrode (dotted line) in a phosphate buffersolution (PBS) of pH 7.4. A redox peak was clearly observed at+550/+200 mV vs Ag/AgCl. The redox peak was not observedwhen the CV was recorded for a mere pDPB-coated electrode asshown in Figure 1b (dashed line). This indicates that the redoxpeak originated from the Mn species complexed with pDPB. Theanodic peak at +550 mV corresponded to the oxidation of Mn2+

to Mn3+, whereas the cathodic one at +200 mV correspondedto the reduction of Mn3+ to Mn2+. It was previously reportedfor a MnO2 film-modified CPE system used to study Mnoxidation states which showed a reduction signal at +300 mV.32

In this case, the signal was attributed to formation of loweroxidation state manganese oxides. Above 400 mV, reoxidationof these oxides to MnO2 occurred. The oxides (+400 mV)showed a similar oxidation potential to ours (+550 mV), butthe reduction potential of our system (+200 mV) was differentfrom the oxide system. This indicates that the oxidation ofMn2+ to Mn3+ is similar, but the reduction of oxidized Mnspecies is a little different due to the different coordinationenvironment in our Mn-pDPB complex system. WhenONOO- (46 µM) was added in a 0.1 M phosphate buffersolution at pH 7.4, there was a slight positive shift in thereduction peak, showing the interaction of Mn attached on thepDPB (Figure 1b, bold line). The three possible 3-dimensionalstructures of Mn-pDPB were emulated by ChemDraw Ultra insimulation, and their stabilized molecular energies were calculatedusing Magnetomotive Force (MMF) as shown in Figure 1c. Themost stable molecule was shown to have molecular binding energyof 83.924 kcal/mol corresponding to C sharing a double bond with2 O atoms, each subsequently linked to Mn2+ ion.

The cathodic and anodic peak currents were dependent onthe scan rate (data not shown). The electron transfer rate constant,ks, for this process was determined to be 2.73 s-1 with theLaviron equation,33 which shows a 5-fold enhancement in theONOO- reduction process due to the presence of Mn2+ ioninvolved in electron transfer compared to the previousreports.18,19 The maximum surface coverage of the complexedMn2+ on the pDPB film at the optimized condition wasestimated using the following equation:34

IP ) n2F2νAΓ/4RT

where Ip is the peak current, n is the number of electrons, F isthe Faraday constant, R is the gas constant, T is temperature,ν is the scan rate, A is the area of the electrode, and Γ is thesurface coverage of Mn2+ species. The surface coverage of thecomplexed Mn2+ species was estimated to be (5.01 ± 0.13) ×10-11 mol/cm2 from the oxidation process of Mn2+ to Mn3+.

ESCA Characterization of the Mn-pDPB Complex. Tocharacterize the modified surfaces, ESCA analyses were carriedout as shown in Figure 2. Figure 2a shows the survey spectraobtained for pDPB (dashed line) and Mn-pDPB complex-modified surfaces (solid line). The pDPB-coated surface did notshow any peak for Mn, whereas the Mn-pDPB complex-modifiedsurface showed two Mn2p peaks, indicating that the Mn2+ specieswas present in the Mn-pDPB complex-modified surface.35 TheO1s spectrum shown in Figure 2b for the pDPB-coated surfaceexhibited a peak at 532.0 eV (dashed line), which correspondedto the C-O bond. The peak shifted to a higher energy of 532.6eV (solid line) after complexation. This indicated that the complexformation between Mn2+ and pDPB occurred through theformation of Mn2+-O bonds. The ESCA spectra of Mn2p peaksin Figure 2c for the Mn-pDPB complex-coated surface wererecorded before any redox potential was applied to the electrode.The Mn2p spectrum exhibited two peaks at 641.2 and 652.9 eVwhich corresponded to 2p3/2 and 2p1/2 environments, respec-tively. To identify the oxidation states of Mn species duringthe redox reaction, ESCA spectra were taken for the Mn-pDPB

(31) Sarneski, J. E.; Brzezinski, L. J.; Anderson, B.; Didiuk, M.; Manchanda, R.;Crabtree, R. H.; Brudvig, G. W.; Schulte, G. K. Inorg. Chem. 1993, 32,3265–3269.

(32) Beyene, N. W.; Kotzian, P.; Schachl, K.; Alemu, H.; Turkusic, E.; Copra,A.; Moderegger, H.; Svancara, I.; Vytras, K.; Kalcher, K. Talanta 2004,64, 1151–1159.

(33) Laviron, E. J. Electroanal. Chem. 1979, 101, 19–28.(34) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980.(35) Zaw, M.; Chiswell, B. Talanta 1995, 42, 27–40.

Scheme 1. Fabrication Steps of Mn-pDPB Modified Electrode and Reaction Mechanism Scheme of the Mn-pDTBModified Electrode with ONOO-


complex-modified surface after oxidation at +550 mV and forthe oxidized Mn-pDPB complex modified surface after reduc-tion at +200 mV. As shown in Figure 2d, the Mn 2p3/2 peaksafter oxidation, belonging to the Mn3+-pDPB complex modi-fied surface, appeared at 642.9 and 641 eV, which correspondedto the Mn3+ and Mn2+ species, respectively. After reduction,the Mn 2p1/2 peaks appeared at 654.2 and 653 eV, correspond-ing to the Mn3+ and Mn2+ species, respectively.35 This clearlyshowed that Mn2+ was first oxidized to Mn3+ and then reducedback to Mn2+ after reacting with ONOO-. Thus, the redoxMn2+/Mn3+ couple chemically adsorbed on pDPB was involvedin the ONOO- detection process.

Optimization of Analysis Parameters. To optimize thesensing condition of the ONOO- microbiosensor, the pH effectof the medium and the applied reduction potential were studiedon the basis of the electrochemical reduction of ONOO- withthe Mn-pDPB modified electrode. The pH effect on analyticalsensitivity was studied in the pH range of 4.0-9.0. Theresponse current increased as media pH increased from 4.0 to7.0 and then showed a decrease above pH 7.5 (Figure 3a).However, the current response did not decrease significantlybetween pH 7.0 and 7.5. Since the physiological pH in the brainis considered to be 7.4, the calibration experiments were done atpH 7.4.

The temperature dependency tests were carried out using theMn-pDPB modified electrode at temperatures of 20-60 °C, as

shown in Figure 3b. The biosensor response decreased as thetemperature increased over 25-60 °C. Hence, all subsequentexperiments were performed at the optimal temperature of 25 °C.

The effect of the applied reduction potential on the chrono-amperometric response was also studied for the electroreductionof ONOO- with the Mn-pDPB modified electrode. The currentresponse increased as the applied potential went from 0.6 V toless positive potentials up to 0.2 V, where the maximumresponse was observed. This tied in well with the cyclicvoltammetric results in that the ONOO- showed a reductionpeak at the same value of 0.2 V. The application of morenegative potentials up to -0.2 V showed declining currentresponse (Figure 3c). Therefore, the Mn-pDPB modifiedelectrode was polarized at 0.2 V versus Ag/AgCl in the chrono-amperometric experiments.

Interference Effect and Selectivity. Oxygen, peroxide, orsuperoxide species interfere with ONOO- detection due to theirsimilar molecular size and the fact that they are precursors orbyproducts of interlinked biological processes.1 Thus, there isa need to eliminate these interfering species. Of the ionpermeable polymers, PEI does not allow cations to permeatethrough. In addition, the PEI layer also prevents microelectrodefouling due to nonspecific adsorption of proteins and otherbiological materials present in the brain.20 In order to removeinterference from positively charged species and ensure long-time stability, a thin PEI film was coated onto the Mn-pDPB

Figure 2. ESCA analysis of pDPB-coated (dashed line) and Mn-pDPB complex-modified (solid line) surfaces; (a) survey spectra, (b) O1speaks before (dashed line) and after (solid line) complexation with Mn2+, (c) Mn2p peaks before application of any potential, and (d) Mn2ppeaks of the Mn-pDPB complex-modified surface after oxidation at +550 mV (solid line), oxidized Mn-pDPB complex surface after reductionat +200 mV (dashed line).


surface of the electrode. The selectivity of the Mn-pDPBmodified electrode was evaluated with chronoamperometry inthe presence of oxygen and other reactive oxygen species, suchas hydrogen peroxide and superoxide as shown in Figure 4a.Figure 4a showed a small response to oxygen in the chrono-amperogram where oxygen was present in the PBS solution (whenno purging was performed with nitrogen). There was littleinterference when varying amounts of other compounds such ashydrogen peroxide and superoxide were added to the PBS testsolution as shown in Figure 4a. ONOO- was added in thesolution in increasing amounts (23, 64, and 140 nM), and thecurrent response of the PEI-coated Mn-pDPB modifiedelectrode increased gradually, indicating that the modifiedelectrode can detect ONOO- more effectively than otherspecies. To further confirm the response of ONOO-, inhibitoryexperiments were performed using a chronoamperometrictechnique where a ONOO- scavenger, such as uric acid,1 wasadded after four successive additions of ONOO- standardsolution (data not shown). The response current rose steeplyand then arrived at an increased steady value after eachaddition of ONOO-. However, upon adding uric acid, thecurrent response declined sharply to the baseline value. Thisis because ONOO- was removed from the test solution almostimmediately by uric acid.

Calibration Plot. To calibrate the ONOO- microbiosensorfor in vitro measurements, the chronoamperometric responseof the Mn-pDPB modified electrode was monitored byintroducing varying concentrations of ONOO- standard solu-

tions. Figure 4b (Inset) showed the typical current-time plotsfor the addition of various ONOO- concentrations in a 0.1 M

Figure 3. Optimizations of experimental conditions of the OONO- biosensor; (a) pH, (b) temperature, and (c) applied potential.

Figure 4. (a) Chronoamperomeric measurements for the interfer-ence effects of different compounds with Mn-pDPB complex-modifiedelectrode. (b, inset) Amperometric responses for ONOO- recordedwith Mn-pDPB complex-modified microbiosensor. Applied potentialwas set at 0.2 V versus Ag/AgCl. (b) Calibration plot for ONOO-

recorded with a Mn-pDPB complex-modified microbiosensor.


PBS solution during experiments. The applied potential wasset at 0.2 V for the electroreduction of ONOO- by theMn-pDPB modified electrode. The response current rosesteeply and then arrived at an increased steady value after eachaddition of ONOO-. Ninety-five percent of steady-state currentswere achieved by the Mn-pDPB modified electrode after about15 s. Figure 4b showed the calibration plots of the Mn-pDPBmodified electrode obtained during an experiment. Under opti-mized conditions, the steady-state currents exhibited a linearrelationship with the ONOO- concentration in the range of 2.0× 10-8-5.0 × 10-7 M for experiments. This range is two ordersof magnitude lower than the values in previously reportedelectrochemical methods which employed the tetraaminoph-thalocyanine complex film as a sensing element.17,36 Theelectrode was found to be reusable eight times, and the relativestandard deviation was found to be 3.4%, after five experimentalruns. The linear dependencies of ONOO- concentration gave anequation of ip (µA) ) (0.298 ± 0.16) + (0.157 ± 0.007) [C] (µM),with a correlation coefficient of 0.994. The sensitivity of theONOO- microbiosensor was 0.157 ± 0.007 µA/µM. Thestability of the ONOO- microbiosensor was examined usingfive experimental runs, and the sensitivity of the ONOO-

microbiosensor was maintained at 86% after two months,indicating high sensor stability. The detection limit of ONOO-

was determined to be 1.9 (±0.2) × 10-9 M by the Mn-pDPBmodified electrode based on a five times measurement for thestandard deviation of the blank noise (95% confidence level, k) 3, n ) 5). This was two orders of magnitude lower thanpreviously reported in in vitro ONOO- sensing.37,38 Thus, thehighly sensitive ONOO- microsensor was obtained and usedin experiments.

Response of the ONOO- Microbiosensor in BloodPlasma. To examine the validity of the proposed biosensorfor the real sample applications, the determination of ONOO-

released in rat blood plasma was studied. Healthy plasmasample does not contain ONOO-, so we performed spikeand recovery experiments to examine the applicability of thisONOO- sensor in a rat plasma sample. The calibrationmethod was used to determine ONOO- concentration.Figure 5a shows the amperogram recorded during the additionof a 1.0 mL of blood plasma sample, followed by adding differentconcentrations of a standard solution of ONOO-. The inset ofthe figure shows the corresponding standard addition plot.The linear regression equation was expressed as Ip (µA) )1.57 (±0.02) + 0.16 (±0.04) [ONOO-] (µM), with thecorrelation coefficient of 0.990, and the relative standarddeviation (RSD) was determined to be 5.8%. The averageconcentration of ONOO- from a rat plasma sample (n ) 5)was determined to be 4.52 ± 0.33 µM., which is comparableto the values previously reported.19,35-38 The ONOO- con-centration recovery was between 95% and 98%, which clearlyindicates the potentiality of this ONOO- sensor for detectionin real biological samples.

Cell Culture Sample Analysis. In addition to blood plasmaexperiments, the ONOO- microbiosensor was also used toprobe the concentration change of ONOO- in cultured cells.Figure 5b shows the extracellular signals of rat glioma YPEN-1cells. The present studies showed that ONOO- production byphorbol myristate acetate (PMA)-stimulated cells was inducedby oxidative stress. When the ONOO- microbiosensor wasremoved from the PBS buffer without cells and placed into theHank buffer saline solution (HBSS) containing stimulated cells,a basal level of ONOO- was detected with the chronoampero-metric technique. A current response of 1.2 µA correspondingto 8.0 (±0.5) × 10-8 M ONOO- was elucidated. These datademonstrated that PMA induced cells to secrete ONOO-. Thus,the direct in vitro monitoring of cells for ONOO- related tooxidative stress will be a useful system for biosensor applica-tions such as drug screening.

CONCLUSIONSA peroxynitrite microbiosensor based on manganese ion

(Mn2+) complexed onto the nanostructured conductingpolymer (pDPB) was fabricated for the measurement ofspiked peroxynitrite in rat plasma sample as well as for the

(36) Tsukahara, H.; Ishida, T.; Mayumi, M. Nitric Oxide 1999, 3, 191–198.(37) Lim, C. H.; Dedon, P. C.; Deen, W. M. Chem. Res. Toxicol. 2008, 21, 2134–

2147.(38) Amatore, C.; Arbault, S.; Guille, M.; Lemaitre, F. Chem. Rev. 2008, 108,

2585–2621.

Figure 5. (a) Amperometric responses (inset) and a standardaddition plot (main) obtained in spiked rat plasma samples. (b)Chronoamperogram illustrating the variation of ONOO- concentrationwith time when transferred from cell-free HBSS to HBSS containing107 YPEN-1 cells. (b, inset) Chronoamperograms showing differentONOO- concentrations with time in cell-free HBSS (dashed line) andHBSS containing 107 YPEN-1 cells (solid line).


in vitro peroxynitrite detection stimulated by PMA incultured cells. The present microbiosensor exhibited a widelinear range between 2.0 × 10-8 and 5.0 × 10-7 M with adetection limit of 1.9 (±0.2) × 10-9 M. The microbiosensorwas calibrated for experiments. The biosensor surface canbe easily regenerated. The response time of this microbio-sensor was within 15 s; thus, it can be used to monitor theextracellular fluctuation of peroxynitrite in biological samples.The spiked peroxynitrite concentrations were determinedin rat blood plasma. PMA stimulated cells to releaseperoxynitrite during oxidative stress. Thus, the peroxynitritebiosensor could be an effective tool for monitoring changes

in in vitro extracellular peroxynitrite levels in response tostimulant drug exposure.

ACKNOWLEDGMENTThis research was supported by the Midcareer Researcher

Program through an NRF grant funded by the MEST, S. Korea(Grant No. 20100029128).

Received for review August 1, 2010. Accepted November5, 2010.

AC102041U


electrochemical detection of peroxynitrite using a biosensor...

Documents